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Maria Chiaffarano
BARTEL AND SZOSTAK EXPERIMENT
HOW DID LIFE BEGIN?
FOUR OVERLAPPING STAGES• Scientists used four stages to understand the origin of life
• Stage 1
• Nucleotides and amino acids were produced prior to the existence of cells
• Stage 2
• Nucleotides became polymerized to form RNA and/or DNA and amino acids become polymerized to form proteins
• Stage 3
• Polymers became enclosed in membranes
• Stage 4
• Polymers enclosed in membranes acquired cellular properties
CHEMICAL SELECTION
• What is chemical selection?
• Chemical within a mixture has special advantageous properties
• Properties allow it to increase in amount
• Hypothesis
• The cellular characteristics that exist today evolved from an “RNA world”
RNA WORLD
• Scientists believe that the world used to consist of RNA based organisms
• Studied the building blocks
• Amino acids and nucleotides
KEY FUNCTIONS OF RNA
• RNA has three key functions that encourage scientists to favor it as the first macromolecule found in protobionts
• Ability to store information in its nucleotides
• Nucleotide sequence has the capacity for self-replication
• RNA has many enzymatic functions
• Act as ribozymes
ADDITIONAL ADVANTAGES OF RNA
• DNA and proteins are not as versatile as RNA
• DNA has limited catalytic activity
• Proteins do not undergo self-replication
• However:
• RNA can perform functions that are characteristics of proteins while simultaneously serving as genetic material with replicative and informational functions
BARTEL AND SZOSTAK EXPERIMENT
• David Bartel and Jack Szostak
• First study that used RNA molecules with a particular function (1993)
• Synthesized a mixture of 10^15 RNA molecules also known as “long RNA”
• First region (5’ end) constant region among all “long RNAs” (identical)
• Second region, variable region (220 nucleotides)
• Hypothesized that the variable region could possibly result in long RNA with the enzymatic ability to catalyze a covalent bond between two adjacent nucleotides
MATERIALS
• Many copies of short RNA
• Had a tag sequence that binds tightly to “beads” (column packing material)
• Had a complementary sequence to a site in the constant region of the long RNA
• No variable region, all the same
• Long RNAs with the constant and variable regions
• Variable regions were made using a PCR step (caused mutations in the region)
OVERVIEW OF THE EXPERIMENT
STEP 1
• The long RNAs and short RNAs were incubated together
• Promote hydrogen bonding
• Time was given for the molecules to form covalent connections (only if the RNA had the enzymatic ability to form covalent bonds)
• The long RNA molecule variable regions may rarely have enzymatic ability to connect the 3’end of short RNA to 5’end of long RNA
STEP 2
• The mixture passed through a column of beads
• The beads would bind to the tag sequence of the short RNA only!
• Tag sequence promoted the binding of the short RNA to the beads
• IF the long RNA had the ability to bind to the short RNA, it would also be attached to the beads
• Additional liquid was added to filter out the long RNAs that did not covalently bond to the short RNAs
STEP 3
• The main purpose of this step was to get rid of the beads
• Low pH solution was added in order to prevent the tag sequence from binding to the beads
• The tightly bound RNAs are flushed out of the column
• They were flushed into a flask and labeled “Pool #1”
• Beads were left behind
STEP 4
• Pool #1 was used to make a second batch of long RNA molecules
• Polymerase chain reaction (PCR) was used
• Variable regions were derived from the variable regions of pool #1 RNA molecules, expected to have enzymatic activity
• Reverse transcriptase was used to make cDNA
• PCR primers recognized beginning and end of the long RNA sequence and copied only this region
• cDNA used as a template to make long RNA via RNA polymerase
STEPS 5 & 6
• The procedure was repeated in order to generate 10 consecutive pools of RNA molecules
• A sample of the original population (Pool #1) and each of the following 10 pools were collected in order to test for the enzymatic ability to catalyze a covalent bond between adjacent nucleotides
CONCLUSION
• Data:
• Chemical selection is possible
• Scientists knew this because the experiment showed that there was an increase in covalent bond formation from pool 1 to pool 10
• In each generation, the catalytic enzyme activity increased
• Pool #10’s enzymatic activity was approximately 3 million times higher than the original random pool of molecules